A spatiotemporally resolved single-cell atlas of the Plasmodium liver stage.

Malaria infection involves an obligatory, yet clinically silent liver stage1,2. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis3, but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing4 and single-molecule transcript imaging5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These 'abortive hepatocytes', which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte.

Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.

Competition for hosts modulates vast antigenic diversity to generate persistent strain structure in Plasmodium falciparum.

In their competition for hosts, parasites with antigens that are novel to the host immune system will be at a competitive advantage. The resulting frequency-dependent selection can structure parasite populations into strains of limited genetic overlap. For the causative agent of malaria, Plasmodium falciparum, the high recombination rates and associated vast diversity of its highly antigenic and multicopy var genes preclude such clear clustering in endemic regions. This undermines the definition of strains as specific, temporally persisting gene variant combinations. We use temporal multilayer networks to analyze the genetic similarity of parasites in both simulated data and in an extensively and longitudinally sampled population in Ghana. When viewed over time, populations are structured into modules (i.e., groups) of parasite genomes whose var gene combinations are more similar within than between the modules and whose persistence is much longer than that of the individual genomes that compose them. Comparison to neutral models that retain parasite population dynamics but lack competition reveals that the selection imposed by host immunity promotes the persistence of these modules. The modular structure is, in turn, associated with a slower acquisition of immunity by individual hosts. Modules thus represent dynamically generated niches in host immune space, which can be interpreted as strains. Negative frequency-dependent selection therefore shapes the organization of the var diversity into parasite genomes, leaving a persistence signature over ecological time scales. Multilayer networks extend the scope of phylodynamics analyses by allowing quantification of temporal genetic structure in organisms that generate variation via recombination or other non-bifurcating processes. A strain structure similar to the one described here should apply to other pathogens with large antigenic spaces that evolve via recombination. For malaria, the temporal modular structure should enable the formulation of tractable epidemiological models that account for parasite antigenic diversity and its influence on intervention outcomes.

Optimal immune specificity at the intersection of host life history and parasite epidemiology.

Hosts diverge widely in how, and how well, they defend themselves against infection and immunopathology. Why are hosts so heterogeneous? Both epidemiology and life history are commonly hypothesized to influence host immune strategy, but the relationship between immune strategy and each factor has commonly been investigated in isolation. Here, we show that interactions between life history and epidemiology are crucial for determining optimal immune specificity and sensitivity. We propose a demographically-structured population dynamics model, in which we explore sensitivity and specificity of immune responses when epidemiological risks vary with age. We find that variation in life history traits associated with both reproduction and longevity alters optimal immune strategies-but the magnitude and sometimes even direction of these effects depends on how epidemiological risks vary across life. An especially compelling example that explains previously-puzzling empirical observations is that depending on whether infection risk declines or rises at reproductive maturity, later reproductive maturity can select for either greater or lower immune specificity, potentially illustrating why studies of lifespan and immune variation across taxa have been inconclusive. Thus, the sign of selection on the life history-immune specificity relationship can be reversed in different epidemiological contexts. Drawing on published life history data from a variety of chordate taxa, we generate testable predictions for this facet of the optimal immune strategy. Our results shed light on the causes of the heterogeneity found in immune defenses both within and among species and the ultimate variability of the relationship between life history and immune specificity.

Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

Bovine tuberculosis disturbs parasite functional trait composition in African buffalo.

Novel parasites can have wide-ranging impacts, not only on host populations, but also on the resident parasite community. Historically, impacts of novel parasites have been assessed by examining pairwise interactions between parasite species. However, parasite communities are complex networks of interacting species. Here we used multivariate taxonomic and trait-based approaches to determine how parasite community composition changed when African buffalo (Syncerus caffer) acquired an emerging disease, bovine tuberculosis (BTB). Both taxonomic and functional parasite richness increased significantly in animals that acquired BTB than in those that did not. Thus, the presence of BTB seems to catalyze extraordinary shifts in community composition. There were no differences in overall parasite taxonomic composition between infected and uninfected individuals, however. The trait-based analysis revealed an increase in direct-transmitted, quickly replicating parasites following BTB infection. This study demonstrates that trait-based approaches provide insight into parasite community dynamics in the context of emerging infections.

C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence.

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase-deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

Oral combination of eugenol oleate and miltefosine induce immune response during experimental visceral leishmaniasis through nitric oxide generation with advanced cytokine demand.

Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis.

A review on the interactions between dendritic cells, filarial parasite and parasite-derived molecules in regulating the host immune responses.

Lymphatic filariasis (LF) is the second leading cause of parasitic disabilities that affects millions of people in India and several other tropical countries. The complexity of this disease is endorsed by various immunopathological consequences such as lymphangitis, lymphadenitis and elephantiasis. The immune evasion strategies that a filarial parasite usually follows are chiefly initiated with the communication between the invaded parasites and parasite-derived molecules, with the Toll-like receptors (TLRs) present on the surface of the antigen-presenting cells (APCs). Classically, the filarial parasites interact with the DCs resulting in lowering of CD4+ T-cell responses. These CD4+ T-cell responses are the key players behind the immune-mediated pathologies associated with LF. In chronic stage, the canonical pro-inflammatory immune responses are shifted towards an anti-inflammatory subtype, which is favouring the parasite survivability within the host. The central theme of this review article is to present the overall immune response elicited when an APC, particularly a DC, encounters a filarial parasite.

Antibody effector functions in malaria and other parasitic diseases: a few needles and many haystacks.

Many parasitic infections stimulate antibody responses in their mammalian hosts. The ability of these antibodies to protect against disease varies markedly. Research has revealed that functional properties of antibodies determine their role in protection against parasites. Investigations of antibodies against Plasmodium spp. have demonstrated a variety of functional activities, ranging from invasion inhibition and parasite growth inhibition to antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity. These activities have been demonstrated with a large variety of parasite molecules at multiple life cycle stages, highlighting the importance of functional antibody responses in malaria. Other parasitic infections have not yet been investigated in similar detail, but these mechanisms are likely to operate in nonmalarial parasitic infections as well. In this report, we review data on the role of functional antibody responses in protection from parasitic infections, highlighting discoveries in malaria, a parasite for which our knowledge base is the most advanced.

NOD1 and NOD2: Beyond Peptidoglycan Sensing.

NOD1 and NOD2 are pattern recognition receptors of the innate immune system with well-established roles in sensing fragments of bacterial peptidoglycan. In addition to their role as microbial sensors, recent evidence indicates that nucleotide-binding oligomerization domains (NODs) can also recognize a broader array of danger signals. Indeed, recent work has expanded the roles of NOD1 and NOD2 to encompass not only sensing of infections with viruses and parasites but also perceiving perturbations of cellular processes such as regulation of the actin cytoskeleton and maintenance of endoplasmic reticulum homeostasis. This review will comment on recent progress and point out emerging questions in these areas.

Inflammatory bowel diseases, the hygiene hypothesis and the other side of the microbiota: Parasites and fungi.

This review tackles the concept of the evolutionary mismatch, in relation with the reduction of the prevalence of the so-called "dirty old friends". These formed the variegated community of parasites and microorganisms, either prokaryotic or eukaryotic, that, over long evolutionary times, co-evolved with humans and their ancestors, inhabiting their digestive tracts, and other body districts. This community of microbial symbionts and metazoan parasites is thought to have evolved a complex network of inter-independence with the host, in particular in relation with their immune stimulating capacity, and with the consequent adaptation of the host immune response to this chronic stimulation. Strictly related to this evolutionary mismatch, the hygiene hypothesis, proposed by David Strachan in 1989, foresees that the increase in the incidence of inflammatory and autoimmune disorders during the twentieth century has been caused by the reduced exposure to parasites and microorganisms, especially in industrialized countries. Among these pathologies, inflammatory bowel diseases (IBDs) occupy a prominent role. From these premises, this review summarizes current knowledge on how variations in the composition of the gut bacterial microbiota, as well as its interactions with fungal communities, influence the overall immune balance, favouring or counteracting gut inflammation in IBDs. Additionally, the effect of worm parasites, either directly on the immune balance, or indirectly, through the modulation of bacterial and fungal microbiota, will be addressed. Finally, we will review a series of studies related to the use of molecules derived from parasitic worms and fungi, which hold the potential to be developed as postbiotics for the treatment of IBDs.

Collectins: Innate Immune Pattern Recognition Molecules.

Collectins are collagen-containing C-type (calcium-dependent) lectins which are important pathogen pattern recognising innate immune molecules. Their primary structure is characterised by an N-terminal, triple-helical collagenous region made up of Gly-X-Y repeats, an a-helical coiled-coil trimerising neck region, and a C-terminal C-type lectin or carbohydrate recognition domain (CRD). Further oligomerisation of this primary structure can give rise to more complex and multimeric structures that can be seen under electron microscope. Collectins can be found in serum as well as in a range of tissues at the mucosal surfaces. Mannanbinding lectin can activate the complement system while other members of the collectin family are extremely versatile in recognising a diverse range of pathogens via their CRDs and bring about effector functions designed at the clearance of invading pathogens. These mechanisms include opsonisation, enhancement of phagocytosis, triggering superoxidative burst and nitric oxide production. Collectins can also potentiate the adaptive immune response via antigen presenting cells such as macrophages and dendritic cells through modulation of cytokines and chemokines, thus they can act as a link between innate and adaptive immunity. This chapter describes the structure-function relationships of collectins, their diverse functions, and their interaction with viruses, bacteria, fungi and parasites.

Unlocking the genetic basis of monarch butterflies' use of medicinal plants.

If there was any doubt of the primary role that plant secondary metabolites play in host-parasite co-evolution, the "From the Cover" paper by Tan et al. (2019) featured in this issue of Molecular Ecology will lay these doubts to rest. The group's previous work on monarch butterflies (Danaus plexippus) infected with the protozoan pathogen Ophryocystis elektroscirrha (OE) demonstrated higher survival and lower spore load on high cardenolide-producing milkweed (Asclepias curassavica) (Figure 1a) compared with low cardenolide-producing milkweed (A. incarnata) (de Roode, Pedersen, Hunter, & Altizer, 2008) (Figure 1b). The mechanism of this protective effect is not directly clear, but a leading hypothesis is that the cardenolides confer protection through toxicity to the parasite. However, the role of the caterpillar immune system in managing this parasite is largely unknown. Novel insights into the influence of toxic plant metabolites on caterpillar immunity are explored in Tan et al. (2019). Using transcriptomics to probe this model system, the authors found that herbivore immune genes were down-regulated and detoxification genes were up-regulated when larvae were reared on the milkweed species with high cardenolide concentrations (A. curassavica). Surprisingly, immune genes were not significantly up- or down-regulated in response to protozoan infection alone. This tantalizing result suggests that sequestered plant metabolites, not immunity, is reining in protozoan infections in these larvae, and promoting survival. As the authors point out, the strategy to invest in sequestration may come at a cost, which is to the detriment of the immune response (Smilanich, Dyer, Chambers, & Bowers, 2009). However, the cost becomes worth the investment when chemical sequestration takes on an antipathogen role. The novelty of the Tan et al. (2019) paper is that they show the investment in sequestration leading to a possible divestment in immunity.

Tinkering with targeting nucleotide signaling for control of intracellular Leishmania parasites.

Nucleotides are one of the most primitive extracellular signalling molecules across all phyla and regulate a multitude of responses. The biological effects of extracellular nucleotides/sides are mediated via the specific purinergic receptors present on the cell surface. In mammalian system, adenine nucleotides are the predominant nucleotides found in the extracellular milieu and mediate a constellation of physiological functions. In the context of host-pathogen interaction, extracellular ATP is recognized as a danger signal and potentiates the release of pro-inflammatory mediators from activated immune cells, on the other hand, its breakdown product adenosine exerts potential anti-inflammatory and immunosuppressive actions. Therefore, it is increasingly apparent that the interplay between extracellular ATP/adenosine ratios has a significant role in coordinating the regulation of the immune system in health and diseases. Several pathogens express ectonucleotidases on their surface and exploit the purinergic signalling as one of the mechanisms to modulate the host immune response. Leishmania pathogens are one of the most successful intracellular pathogens which survive within host macrophages and manipulate protective Th1 response into disease promoting Th2 response. In this review, we discuss the regulation of extracellular ATP and adenosine levels, the role of ATP/adenosine counter signalling in regulating the inflammation and immune responses during infection and how Leishmania parasites exploit the purinergic signalling to manipulate host response. We also discuss the challenges and opportunities in targeting purinergic signalling and the future prospects.

Parasites and allergy: Observations from Brazil.

Brazil is a middle-income country undergoing the epidemiological transition. Effects of changes in daily life habits and access to clean water, sanitation and urban services on a growing urban population have contributed to a double burden of both infectious and noncommunicable chronic diseases. Studies have indicated that parasite infections may modulate the human immune system and influence the development of allergic conditions such as asthma. However, there is no consensus in the published literature on the effects of parasitic infections on allergy, perhaps as a consequence of factors determining the epidemiology of these infections that vary between populations such as age of first infection, duration and chronicity of infections, parasite burden and species, and host genetic susceptibility. In this review, we discuss the observations from Brazil concerning the relationship between parasite infections and allergy.

Molecular characterization and expression analysis of T cell receptor (TCR) γ and δ genes in dojo loach (Misgurnus anguillicaudatus) in response to bacterial, parasitic and fungal challenge.

In mammalian, T-cell receptors (TCRs) play a key role in recognizing the presented antigen from external to protect organisms against environmental pathogens. To understand the potential roles of TCRγ and TCRδ in dojo loach (Misgurnus anguillicaudatus), Ma-TCRγ and Ma-TCRδ cDNAs were cloned and their gene expression profiles were investigated after bacterial, parasitic and fungal challenge. The open reading frame (ORF) of Ma-TCRγ and Ma-TCRδ cDNAs contained 948 and 867 bp, encoding 316 and 288 amino acid residues, respectively. Structurally, Ma-TCRγ and Ma-TCRδ were consisted of a signal peptide, a variable region, a constant region (IgC), a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic domain (CYT), which were similar to those of other vertebrates. Multiple sequence alignment and phylogenetic analysis showed Ma-TCRγ and Ma-TCRδ were closely related to fish of Cyprinidae family. Ma-TCRγ and Ma-TCRδ were widely expressed in all tested organs/tissues, as the highest expressions of Ma-TCRγ and Ma-TCRδ were detected in kidney and gill, respectively. In addition, three infection models of dojo loach with bacteria (F. columnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia sp.) were constructed. The morphological changes of gills and skin after challenged with F. columnare G4 and Ichthyophthirius multifiliis were investigated. Compared to F. columnare G4 infection, mRNA expression of both TCRγ and TCRδ showed higher sensitivity in classical immune organs (kidney and spleen) and mucosal tissues (skin and gill) after challenge with Ichthyophthirius multifiliis and Saprolegnia sp. Our results first indicated that TCRγ and TCRδ of dojo loach might function differently in response to challenge with different pathogens.

Parasites and epilepsy: Understanding the determinants of epileptogenesis.

There is a large body of evidence suggesting that parasites could be a major preventable risk factor for epilepsy in low- and middle-income countries. We review potentially important substrates for epileptogenesis in parasitic diseases. Taenia solium is the most widely known parasite associated with epilepsy, and the risk seems determined mainly by the extent of cortical involvement and the evolution of the primary cortical lesion to gliosis or to a calcified granuloma. For most parasites, however, epileptogenesis is more complex, and other favorable host genetic factors and parasite-specific characteristics may be critical. In situations where cortical involvement by the parasite is either absent or minimal, parasite-induced epileptogenesis through an autoimmune process seems plausible. Further research to identify important markers of epileptogenesis in parasitic diseases will have huge implications for the development of trials to halt or delay onset of epilepsy.

Glycans in the roles of parasitological diagnosis and host-parasite interplay.

The investigation of the glycan repertoire of several organisms has revealed a wide variation in terms of structures and abundance of glycan moieties. Among the parasites, it is possible to observe different sets of glycoconjugates across taxa and developmental stages within a species. The presence of distinct glycoconjugates throughout the life cycle of a parasite could relate to the ability of that organism to adapt and survive in different hosts and environments. Carbohydrates on the surface, and in excretory-secretory products of parasites, play essential roles in host-parasite interactions. Carbohydrate portions of complex molecules of parasites stimulate and modulate host immune responses, mainly through interactions with specific receptors on the surface of dendritic cells, leading to the generation of a pattern of response that may benefit parasite survival. Available data reviewed here also show the frequent aspect of parasite immunomodulation of mammalian responses through specific glycan interactions, which ultimately makes these molecules promising in the fields of diagnostics and vaccinology.

B-1 cell response in immunity against parasites.

The peritoneal cavity has a microenvironment capable of promoting proliferation, differentiation, and activation of the resident cells and recruitment of blood cells through the capillary network involved in the peritoneum. Among the cells found in the peritoneal cavity, B-1 cells are a particular cell type that contains features that are not very well defined. These cells differ from conventional B lymphocytes (B-2) by phenotypic, functional, and molecular characteristics. B-1 cells can produce natural antibodies, migrate to the inflammatory focus, and have the ability to phagocytose pathogens. However, the role of B-1 cells in immunity against parasites is still not completely understood. Several experimental models have demonstrated that B-1 cells can affect the susceptibility or resistance to parasite infections depending on the model and species. Here, we review the literature to provide information on the peculiarities of B-1 lymphocytes as well as their interaction with parasites.